High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader

Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP, resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.     

CHORAL: a differential geometry approach to the prediction of the cores of protein structures

MOTIVATION: Although the cores of homologous proteins are relatively well conserved, amino acid substitutions lead to significant differences in the structures of divergent superfamilies. Thus, the classification of amino acid sequence patterns and the selection of appropriate fragments of the protein cores of homologues of known structure are important for accurate comparative modelling. RESULTS: CHORAL utilizes a knowledge-based method comprising an amalgam of differential geometry and pattern recognition algorithms to identify conserved structural patterns in homologous protein families. Propensity tables are used to classify and to select patterns that most likely represent the structure of the core for a target protein. In our benchmark, CHORAL demonstrates a performance equivalent to that of MODELLER.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Role of non-quantum acetylcholine secretion in neural control of the membrane potential in skeletal muscle fibers of rats

Experiments on muscle fibers of the rat diaphragm (in vitro denervation) showed that their three-hour incubation in the cultural medium results in an 8-mV drop in the resting membrane potential (RMP). Addition of 5·10^–8 M carbacholine to the cultural medium, mimicing the effect of non-quantum acetylcholine, delayed depolarization of the denervated muscle. The effect of carbacholine could not be eliminated byd-tubocurarine (5·10^–6 M), a postsynaptic acetylcholine receptor blocker, and by ouabain (1·10^–4 M), and inhibitor of Na⁺, K⁺-ATPase of the membrane. At the same time, the effect could be completely eliminated by Mg²⁺ ions (5·10^–3 M), which blocked Ca²⁺ channels of the membrane, by N-nitroarginine (1·10^–4 M), which inhibited the enzyme NO-synthase, and by hemoglobin (2·10^–5 M), which inactivated the extracellular NO molecules. It is concluded that the released non-quantum acetylcholine can contribute to neural control of RMP of cross-striated muscle fibers via the Ca²⁺-dependent activation of NO synthesis in the sarcoplasm. The NO molecules can play the role of a retrograde signal indicative of the normal functioning of the neuromuscular synapse. The impairment of this link caused by a denervation-induced cessation of the non-quantum secretion can serve as a signal triggering the early changes in the muscle membrane following nerve transection.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 67–71, January–February, 1995.     

Field-induced reorganization of the neural membrane lipid bilayer: a proposed role in the regulation of ion-channel dynamics

We present a computational model demonstrating that an electric field propagating in the plane of the neural membrane during transmembrane ion movement creates lateral concentration gradients of the lipids. Due to this field-induced reorganization, ethenes of the lipid chains become aligned and polarized. This finding is interpreted within the context of molecular studies of protein folding in biological membranes. We propose that electrostatic interactions between membrane dipoles and charged amino acid residues of the unfolded ion-channel protein regulate protein-folding kinetics (channel closing). These electrostatic interactions thus regulate electrical signaling in neurons.     

Assembling neurospheres: dynamics of neural progenitor/stem cell aggregation probed using an optical trap

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (～5 s) and most probable minimum distance of approach (4-6 μm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (～18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and "stemness". Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.     

Developmental control of cell morphogenesis: a focus on membrane growth

To date, the role of transport and insertion of membrane in the control of membrane remodelling during cell and tissue morphogenesis has received little attention. In contrast, the contributions of cytoskeletal rearrangements and both intercellular and cell-substrate attachments have been the focus of many studies. Here, we review work from many developmental systems that highlights the importance of polarized membrane growth and suggests a general model for the role of endocytic recycling during cell morphogenesis. We also address how the spatio-temporal control of membrane insertion during development can account for various classes of tissue rearrangements. We suggest that tubulogenesis, tissue spreading and cell intercalation stem mostly from a remarkably small number of cell intrinsic surface remodelling events that confer on cells different modes of migratory behaviours.     

Electro-permeabilization of cell membranes: effect of the resting membrane potential

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.     

Mitochondrial membrane potential and nucleosidic inhibitors of HIV reverse transcriptase: a cytometric approach

Mitochondrial toxicity is a relevant side effect of anti-HIV antiretroviral therapy. Adequate experimental models and advanced technologies are crucial to investigate properly mitochondrial toxicity. Functional flow cytometry allows a rapid and sensitive evaluation of several parameters on single cells, and is an excellent tool to investigate the impact of antiviral drug on mitochondrial activity. We used such approach to analyze in vitro mitochondrial toxicity induced by stavudine and zidovudine on cell lines of different origin (hemopoietic: A301, U937, CEM, K562; hepatic: HepG2), and found that the cell lines had a different sensitivity to the action of the drugs.     

GPI-anchor remodeling: potential functions of GPI-anchors in intracellular trafficking and membrane dynamics

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. GPI is synthesized and transferred to proteins in the endoplasmic reticulum. GPI-anchored proteins are then transported from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. GPI-anchor functions as a sorting signal for transport of GPI-anchored proteins in the secretory and endocytic pathways. After GPI attachment to proteins, the structure of the GPI-anchor is remodeled, which regulates the trafficking and localization of GPI-anchored proteins. Recently, genes required for GPI remodeling were identified in yeast and mammalian cells. Here, we describe the structural remodeling and function of GPI-anchors, and discuss how GPI-anchors regulate protein sorting, trafficking, and dynamics. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Polysialylation increases lateral diffusion of neural cell adhesion molecule in the cell membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.     

Role of calcium ions and spike activity in the neurotrophic control of membrane potential in rat muscle fibers

Blockade of calcium permeability produced an increase in postdenervation depolarization of rat diaphragm muscle fibers during in vitro experiments, while increased Ca²⁺ concentration in the sarcoplasm induced by caffeine led to hyperpolarization of the muscle membrane. Direct stimulation of the muscles or carbamylcholine application retarded the reduction of membrane potential in the muscle fibers. Verapamil and d-tubocurarine eliminated the hyperpolarizing effect of stimulation. The hyperpolarizing effects of carbamylcholine applied in conjunction with stimulation did not produce an accumulated action on the membrane. It is deduced that the factors governing membrane potential in the muscle fibers are acetylcholine and Ca²⁺ reaching the sarcoplasm mainly through the acetylcholine-sensitive ionic channels during the process of nerve impulse fluxes.S. V. Kurashov Medical Institute, Kazan', Ministry of Public Health of the RSFSR. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 449–456, July–August, 1987.     

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk

Coordination between functionally related adjacent tissues is essential during development. For example, formation of trunk neural crest cells (NCCs) is highly influenced by the adjacent mesoderm, but the molecular mechanism involved is not well understood. As part of this mechanism, fibroblast growth factor (FGF) and retinoic acid (RA) mesodermal gradients control the onset of neurogenesis in the extending neural tube. In this paper, using gain- and loss-of-function experiments, we show that caudal FGF signaling prevents premature specification of NCCs and, consequently, premature epithelial-mesenchymal transition (EMT) to allow cell emigration. In contrast, rostrally generated RA promotes EMT of NCCs at somitic levels. Furthermore, we show that FGF and RA signaling control EMT in part through the modulation of elements of the bone morphogenetic protein and Wnt signaling pathways. These data establish a clear role for opposition of FGF and RA signaling in control of the timing of NCC EMT and emigration and, consequently, coordination of the development of the central and peripheral nervous system during vertebrate trunk elongation.     

Marked changes in red cell membrane proteins in hereditary spherocytosis: a proteomics approach

Hereditary spherocytosis (HS) is the most common red cell membrane defect resulting from protein abnormalities. However, changes in red cell membrane proteins in HS remain under-investigated. We therefore evaluated red cell membrane proteome in non-splenectomized, mild-degree HS patients (n = 9) compared to healthy individuals (n = 5). Proteins derived from the red cell membranes of each subject were resolved in each two-dimensional gel and visualized by Deep Purple fluorescence staining. Spot matching and quantitative intensity analysis revealed 56 differentially expressed protein spots (41 increased and 15 decreased), which were then successfully identified by quadrupole time-of-flight mass spectrometry. Among these, seven isoforms/subunits of spectrin were markedly increased (up to 10.51 folds), whereas two isoforms/subunits of band-3 protein were decreased approximately 50% as compared to normal red cells. However, two isoforms/subunits of protein 4.1 were increased, while another isoform/subunit was decreased. All these significantly altered proteins were subjected to global protein network analysis using Ingenuity Pathways Analysis tool, which revealed three important networks related to HS, including Network I: Cell death, genetic and hematological disorders; Network II: Cell cycle, carbohydrate metabolism and molecular transport; and Network III: Genetic and hematological disorders, cell-to-cell signaling and interactions. These data offer many opportunities and new roadmaps for further functional studies to better understand the biology and pathogenic mechanisms of HS.     

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.     

Identification of plasma-responsive outer membrane proteins and their vaccine potential in Edwardsiella tarda using proteomic approach

We have used differential sub-proteomic methodologies to detect Edwardsiella tarda outer membrane (OM) protein expression regulation during interaction with fish and human plasma, which is the critical step of the bacterial invasion internal organs via blood circulation. Seven and nine OM proteins were differentially expressed in response to fish and human plasma stress, respectively. Six proteins, TolB2, ETAE_2935, ETAE_0245, EvpA, ETAE_2675 and OmpA, were the shared proteins with the similar changes between the two plasma treatments. Except for EvpA, which was a known protein involved in bacterial pathogenesis and stress sensing, the others were first reported here to be related to bacterial invasion and infection. Out of them, four, upregulated ETAE_0245 and OmpA and downregulated ETAE_2675 and ETAE_2935, were selected for investigation of immune protection. The upregulated OmpA and ETAE_0245 were able to induce bactericidal antibodies in mice. These findings demonstrate that differential proteomic methodologies following protein expression regulation to interaction between host and pathogen with bacterial challenge post immunization of these altered proteins is a valid approach for identifying new vaccine candidates and nicely complements other high throughput mining strategies used for vaccine discovery.